Compositions and methods for suppressing fibrocytes

ABSTRACT

The present invention relates to the ability of anti-FcγR antibodies to suppress fibrocytes. Methods and compositions for suppressing fibrocytes are provided. These methods are useful in a variety of applications including treatment and prevention of conditions resulting from fibrosis in the liver, kidney, lung, heart and pericardium, eye, skin, mouth, pancreas, gastrointestinal tract, brain, breast, bone marrow, bone, genitourinary system, a tumor, or a wound.

PRIORITY CLAIM

The present application is a continuation-in-part under 35 U.S.C. §120of PCT patent application serial number PCT/US2006/005229, filed Feb.15, 2006 and titled “Methods and Conditions for Suppressing FibrocyteDifferentiation” and Ser. No. 11/158,966, now U.S. Pat. No. 7,666,432U.S. Application No. 2005/0238620 filed Jun. 22, 2005, which is acontinuation-in-part under 35 U.S.C. §120 of PCT patent applicationserial number PCT/US2003/040957, filed Dec. 22, 2003 and titled “Methodsand Conditions for Suppressing Fibrocyte Differentiation”, published inEnglish as WO 2004/058292 on Jul. 15, 2004; which claims priority to thefollowing: U.S. Provisional Patent Applications 60/436,046, filed Dec.23, 2002; U.S. 60/436,027, filed Dec. 23, 2002; U.S. 60/515,776, filedOct. 30, 2003; U.S. 60/519,467, filed Nov. 12, 2003; and U.S. 60/525,175filed Nov. 26, 2003. Pertinent parts of the application are incorporatedby reference herein.

FIELD OF THE INVENTION

The present invention relates to the ability of anti-FcγR antibodies,aggregated IgG, and/or cross-linked IgG to suppress fibrocytes.Accordingly, it may include compositions and methods for suppressingfibrocytes. These compositions and methods may be useful in a variety ofapplications, for example, those in which decreased fibrocyte formationis beneficial, such as treatment of fibrosing diseases and asthma.

BACKGROUND

Fibrocytes

Inflammation is the coordinated response to tissue injury or infection.The initiating events are mediated by local release of chemotacticfactors, platelet activation, and initiations of the coagulation andcomplement pathways. These events stimulate the local endothelium,promoting the extravasation of neutrophils and monocytes. The secondphase of inflammation is characterized by the influx into the tissue ofcells of the adaptive immune system, including lymphocytes. Thesubsequent resolution phase, when apoptosis of the excess leukocytes andengulfment by tissue macrophages takes place, is also characterized byrepair of tissue damage by stromal cells, such as fibroblasts.

In chronic inflammation, the resolution of inflammatory lesions isdisordered, with the maintenance of inflammatory cells, fibroblasthyperplasia, and eventual tissue destruction. The mechanisms that leadto these events are complex, but include enhanced recruitment, survivaland retention of cells and impaired emigration.

The source of fibroblasts responsible for repair of wound lesions or inother fibrotic responses is controversial. The conventional hypothesissuggests that local quiescent fibroblasts migrate into the affectedarea, produce extracellular matrix proteins, and promote woundcontraction or fibrosis. An alternative hypothesis is that circulatingfibroblast precursors (called fibrocytes) present within the bloodmigrate to the sites of injury or fibrosis, where they differentiate andmediate tissue repair and other fibrotic responses.

Fibrocytes are fibroblast-like cells that appear to participate in woundhealing and are present in pathological lesions associated with, interalia, asthma, pulmonary fibrosis and scleroderma. Fibrocytes are knownto differentiate from a CD14+ peripheral blood monocyte precursorpopulation. Fibrocytes may also differentiate from other sources.Fibrocytes express markers of both hematopoietic cells (CD45, MHC classII, CD34) and stromal cells (collagen types I and III and fibronectin).Fibrocytes at sites of tissue injury secrete inflammatory cytokines,extracellular matrix proteins and promote angiogenesis and woundcontraction. Fibrocytes are also associated with the formation offibrotic lesions after infection or inflammation, and are implicated infibrosis associated with autoimmune diseases.

Control of fibrocyte differentiation is likely to be important in thecontrol of many diseases and processes. Fibrocytes are associated with avariety of processes and diseases including scleroderma, keloidscarring, rheumatoid arthritis, lupus, nephrogenic fibrosing dermopathy,and idiopathic pulmonary fibrosis. They play a role in the formation offibrotic lesions after Schistosoma japonicum infection in mice and arealso implicated in fibrosis associated with autoimmune diseases.Fibrocytes have also been implicated in pathogenic fibrosis, fibrosisassociated with radiation damage, Lyme disease and pulmonary fibrosis.CD34+ fibrocytes have also been associated with stromal remodeling inpancreatitis and stromal fibrosis, whereas lack of such fibrocytes isassociated with pancreatic tumors and adenocarcinomas. Fibrosisadditionally occurs in asthma patients and possibly other pulmonarydiseases such as chronic obstructive pulmonary disease when fibrocytesundergo further differentiation into myofibroblasts.

Fibrocytes may also play a role in a variety of conditions, likely evensome in which fibrocyte formation is not currently known. Someadditional conditions may include congestive heart failure, otherpost-ischemic conditions, post-surgical scarring including abdominaladhesions, corneal refraction surgery, and wide angle glaucomatrabeculectomy. Fibrocytes are also implicated in liver fibrosis andcirrhosis. See Tatiana Kisseleva et al, Bone Marrow-Derived FibrocytesParticipate in Pathogenesis of Liver Fibrosis, 45 Journal of Hepatology429-438 (September 2006); see also F. P. Russo et al, The Bone MarrowFunctionality Contributes to Liver Fibrosis, 130(6) Gastroenterology1807-21 (May 2006). Fibrocytes are important in the formation of tumors,particularly stromal tissue in tumors. Recent evidence also suggeststhat fibrocytes may further differentiate into adipocytes and thus playa role in obesity.

It has been previously identified that fibrocytes may differentiate fromCD14+ peripheral blood monocytes, and the presence of human serumdramatically delays this process. The factor in human serum thatinhibits fibrocyte differentiation is serum amyloid P (SAP). SAP, amember of the pentraxin family of proteins that includes C-reactiveprotein (CRP), is produced by the liver, secreted into the blood, andcirculates in the blood as stable pentamers. SAP binds to receptors forthe Fc portion of IgG antibodies (FcγR) on a variety of cells and mayeffectively cross-link FcγR without additional proteins because SAP is apentameric protein with five potential FcγR binding sites per molecule.As SAP binds to FcγR, intracellular signaling events consistent withFcγR activation are initiated.

Anti-FcγR Antibodies

It has also been identified that anti-FcγR antibodies may also preventthe differentiation of peripheral blood monocytes into fibrocytes.Anti-FcγR antibodies are IgG antibodies that bind to receptors for theFc portion of IgG antibodies (FcγR). The anti-FcγR antibodies bindthrough their variable region, and not through their constant (Fc)region. However, IgG from the appropriate source (e.g. human IgG forhuman receptors) may normally bind to FcγR through its Fc region. FcγRare found on the surface of a variety of hematopoietic cells. There arefour distinct classes of FcγR. FcγRI (CD64) is expressed by peripheralblood monocytes and binds monomeric IgG with a high affinity. FcγRII(CD32) and FcγRIII (CD16) are low affinity receptors for IgG and onlyefficiently bind aggregated IgG. FcγRII is expressed by peripheral bloodB cells and monocytes, whereas FcγRIII is expressed by NK cells and asubpopulation of monocytes. FcγRIV was recently identified in mice andis present on murine peripheral blood monocytes and neutrophils,macrophages and dendritic cells and efficiently binds murine IgG2a andIgG2b antibodies. There is a putative human FcγRIV gene, but thebiological function of the protein, such as ligand specificity andcellular expression is, as yet unknown.

Peripheral blood monocytes express both FcγRI and FcγRII (asubpopulation of monocytes express FcγRIII), whereas tissue macrophagesexpress all three classical FcγR. Clustering of FcγR on monocytes byIgG, either bound to pathogens or as part of an immune complex,initiates a wide variety of biochemical events.

FcγR activation and induction of intracellular signaling pathways mayoccur when multiple FcγR are cross-linked or aggregated. This FcγRactivation leads to a cascade of signaling events initiated by two mainkinases. The initial events following FcγR activation involve thephosphorylation of intracellular immunoreceptor tyrosine activationmotifs (ITAMs) present on the cytoplasmic tail of FcγRIIa or the FcR-γchain associated with FcγRI and FcγRIII, by Src-related tyrosine kinases(SRTK). In monocytes, the main Src-kinases associated with FcγRI andFcγRII are hck and lyn. The phosphorylated ITAM then recruit cytoplasmicSH2-containing kinases, especially Syk, to the ITAMs and Syk thenactivates a series of downstream signaling molecules.

Anti-FcγR antibodies for FcγRI (anti-FcγRI) and for FcγRII (anti-FcγRII)are able to bind to either FcγRI or FcγRII, respectively. These FcγR maythen be cross-linked by the binding of additional antibodies or othermeans. This process initiates intracellular signaling events consistentwith FcγR activation.

Scleroderma

Scleroderma is a non-inherited, noninfectious disease that has a rangeof symptoms. It involves the formation of scar tissue containingfibroblasts in the skin and internal organs. The origin of thefibroblasts is unknown. In mild or early cases of scleroderma, there isa hardening of the skin, fatigue, aches and sensitivity to cold. In moresevere and later stages, there is high blood pressure, skin ulcers,difficulty moving joints, and death from lung scarring or kidneyfailure. Approximately 300,000 people in the U.S. have scleroderma. Thedisease has similarities to lupus and rheumatoid arthritis. There is nocure or significant treatment for scleroderma and even diagnosis isdifficult because there is no clinical test.

Nephrogenic Fibrosing Dermopathy

Nephrogenic fibrosing dermopathy (NFD) is a newly recognizedscleroderma-like fibrosing skin condition. It develops in patients withrenal insufficiency. Yellow scleral plaques and circulatingantiphospholipid antibodies have been proposed as markers of NFD. Dualimmunohistochemical staining for CD34 and pro-collagen in the spindlecells of NFD suggest that the dermal cells of NFD may representcirculating fibrocytes recruited to the dermis. Therefore, inhibition offibrocyte formation may alleviate symptoms of this disease.

Asthma

Asthma affects more than 100 million people worldwide, and itsprevalence is increasing. Asthma appears to be caused by chronic airwayinflammation. One of the most destructive aspects of asthma isremodeling of the airways in response to chronic inflammation. Thisremodeling involves thickening of the lamina reticularis (thesubepithelial reticular basement membrane surrounding airways) due tofibrosis. The airway passages then become constricted due to thethickened airway walls.

The thickened lamina reticularis in asthma patients contains abnormallyhigh levels of extracellular matrix proteins such as collagen I,collagen III, collagen V, fibronectin and tenascin. The source of theseproteins appears to be a specialized type of fibroblast calledmyofibroblasts.

In asthma patients, CD34+/collagen I+ fibrocytes accumulate near thebasement membrane of the bronchial mucosa within 4 hours of allergenexposure. 24 hours after allergen exposure, labeled monocytes/fibrocyteshave been observed to express α-smooth muscle actin, a marker formyofibroblasts. These observations suggest that in asthma patientsallergen exposure causes fibrocytes from the blood to enter thebronchial mucosa, differentiate into myofibroblasts, and then causeairway wall thickening and obstruct the airways. Further, there is acorrelation between having a mutation in the regulatory regions of thegenes encoding monocyte chemoattractant protein 1 or TGFβ-1 and theseverity of asthma. This also suggests that recruitment of monocytes andappearance of myofibroblasts lead to complications of asthma.

Thickening of the lamina reticularis distinguishes asthma from chronicbronchitis or chronic obstructive pulmonary disease and is found evenwhen asthma is controlled with conventional medications. An increasedextent of airway wall thickening is associated with severe asthma. Nomedications or treatments have been found to reduce thickening of thelamina reticularis. However, it appears likely that reducing the numberof myofibroblasts found in the airway walls may reduce thickening orhelp prevent further thickening.

Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a unique type of chronicfibrosing lung disease of unknown etiology. The sequence of thepathogenic mechanisms is unknown, but the disease is characterized byepithelial injury and activation, the formation of distinctivesubepithelial fibroblast/myofibroblast foci, and excessive extracellularmatrix accumulation. These pathological processes usually lead toprogressive and irreversible changes in the lung architecture, resultingin progressive respiratory insufficiency and an almost universallyterminal outcome in a relatively short period of time. While researchhas largely focused on inflammatory mechanisms for initiating thefibrotic response, recent evidence strongly suggests that disruption ofthe alveolar epithelium is an underlying pathogenic event. Given therole played by fibrocytes in wound healing and their known role inairway wall thickening in asthma, it appears likely that overproductionof fibrocytes may be implicated in IPF.

SUMMARY

The present invention may include compositions and methods forsuppressing fibrocytes. In the context of the present invention, theterm “suppressing fibrocytes” refers to one or more of inhibiting theproliferation of fibrocytes, inhibiting the development of fibrocytes,including the development or differentiation of a cell into a fibrocyte,and promoting the development or differentiation of fibrocytes intonon-fibrocytic cell types.

In selected embodiments, fibrocytes may be suppressed in a targetlocation by providing anti-FcγR antibodies that are able to cross-linkFcγR. The target location may be located in vitro or in vivo.Specifically, the target location may be located in a mammal, such as ahuman patient.

In vivo, the target location may include an entire organism or a portionthereof and the composition may be administered systemically or it maybe confined to a particular area, such as an organ or tissue.

Suppressing fibrocytes may alleviate symptoms of numerous fibrosingdiseases or other disorders caused by fibrosis. In a specificembodiment, administration of anti-FcγR antibodies may be used to treatthe effects of unwanted fibrocytes. For example, it may be used to treatfibrosis in the liver, kidney, lung, heart and pericardium, eye, skin,mouth, pancreas, gastrointestinal tract, brain, breast, bone marrow,bone, genitourinary, a tumor, or a wound.

BRIEF DESCRIPTION OF THE DRAWINGS

The following figures form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention.

FIG. 1 shows the effects of cross-linked and non-cross-linked anti-FcγRantibodies on fibrocyte differentiation from Peripheral BloodMononuclear Cells (PBMC). PBMC at 2.5×10⁵ cells per ml were cultured inserum-free medium for 5 days in the presence or absence of 1 μg/ml ofthe indicated F(ab′)₂ anti-FcγR or control IgG1 antibodies, in thepresence (black bars) or absence (white bars) of 500 ng/ml goat F(ab′)₂anti-mouse IgG, which cross-links the F(ab′)₂. Cells were thenair-dried, fixed, stained, and fibrocytes were enumerated by morphology.

FIG. 2 shows the effects of SRTK and Syk inhibitors on the ability ofanti-FcγR antibodies on fibrocyte differentiation from PBMC. PBMC wereincubated for 60 minutes at 4° C. with 10 nM PP2, PP3, or Syk inhibitor.PBMC at 2.5×10⁵ cells per ml were then cultured in serum-free medium for5 days in the presence or absence of 1 μg/ml of the indicated murineF(ab′)₂ anti-FcγR antibodies, in the presence or absence of 500 ng/mlgoat F(ab′)₂ anti-mouse IgG. Results are expressed as the mean±SD of thenumber of fibrocytes per 2.5×10⁵ cells (one of two separate donors).

FIG. 3 shows the effects of FcγR aggregation and the effects of SRTK andSyk on fibrocyte differentiation from monocytes. PBMC were at 2.5×10⁵cells per ml were incubated for 60 minutes at 37° C. Non-adherent cellswere then removed by pipetting, resulting in a substantially monocytecell sample. The adherent monocytes were incubated for 60 minutes at 4°C. in the presence or absence of 10 nM PP2, PP3 or Syk inhibitor.Monocytes were then washed twice and cultured in the presence or absenceof heat-aggregated human IgG for 60 minutes at 4° C. This IgG was not ananti-FcγR IgG, but instead was able to bind through its Fc region. Themonocytes were then washed twice, and the non-adherent cells werereplaced to a final concentration of 2.5×10⁵ cells per ml and thencultured for 5 days at 37° C. in serum-free medium. Results areexpressed as the mean±SEM of the number of fibrocytes per 2.5×10⁵ cells(n=3 separate donors.

FIG. 4 shows the effects of cross-linking IgG and other antibodyisotypes and on fibrocyte differentiation.

In FIG. 4A, PBMC were incubated with the indicated concentrations ofmonomeric human IgG for 60 minutes. PBMC were then washed and incubatedin the presence (white boxes) or absence (black boxes) of 500 ng/ml goatF(ab′)2 anti-human IgG. PBMC were then cultured at 2.5×10⁵ cells per mlin serum-free medium for 5 days. PBMC were then air-dried, fixed,stained, and fibrocytes were enumerated by morphology. Results areexpressed as the mean±SEM of number of fibrocytes per 2.5×10⁵ cells (n=4separate donors).

In FIG. 4B, PBMC were cultured as in FIG. 4A in the presence of theindicated concentrations of heat-aggregated human IgG or heat-aggregatedhuman F(ab′)₂. Results are expressed as the ±SEM of number of fibrocytesper 2.5×10⁵ cells (n=3 separate donors).

In FIG. 4C, PBMC were cultured as in FIG. 4A in the presence of 20 μg/mlof native or heat-aggregated human IgA, IgE, IgG or IgM.

FIG. 5 shows the effects of monomeric IgG on the ability of SAP to bindto monocytes and inhibit their differentiation. PBMC were cultured inserum-free medium in the presence of a range of concentrations ofmonomeric IgG for 60 minutes. SAP, at the concentrations indicated, wasthen added and the cells were cultured for 4 days.

FIG. 6 shows the effects of ligation and cross-linking of Fc receptorson monocyte to fibrocyte differentiation. Soluble immune complexes(ovalbumin-antibody), particulate immune complexes, including opsonisedsheep red blood cells (SRBC) and heat-aggregated IgG were used. In FIG.6A PBMC cultured for 4 days with ovalbumin or anti-ovalbumin mAb alone,or ovalbumin:anti-ovalbumin immune complexes. FIG. 6B shows the effectsof SRBC alone and SRBC opsonised with rabbit anti-SRBC at 20:1 and 40:1SRBC:monocyte ratios. Finally, FIG. 6C shows the effects on PBMC ofheat-aggregated IgG and heat-aggregated F(ab)₂. Stars in 6A and 6Bindicate statistically significant differences.

FIG. 7 shows the effects of anti-FcγR antibodies on monocytedifferentiation. Stars indicate a statistically significant differencefrom control.

DETAILED DESCRIPTION

The regulation of events leading to fibrosis involves the proliferationand differentiation of fibrocytes. Fibrocytes are a distinct populationof fibroblast-like cells derived from peripheral blood monocytes thatnormally enter sites of tissue injury to promote angiogenesis and woundhealing. Fibrocytes differentiate from CD14+ peripheral blood monocytes,and may differentiate from other PBMC cells. The presence of anti-FcγRantibodies, aggregated IgG, and/or cross-linked IgG may inhibit or atleast partially delay this process.

Compositions containing anti-FcγRI antibodies and/or anti-FcγRIIantibodies, and/or cross-linked or aggregated IgG, which may bind toFcγR through the Fc region, may be used to suppress fibrosis ininappropriate locations and in fibrosing disorders and chronicinflammatory conditions, inter alia.

In specific embodiments, compositions containing approximately 1 μg/mlanti-FcγR antibodies may be effective to inhibit fibrocyte proliferationor differentiation by approximately 50%. In other embodiments,compositions may contain an amount sufficient to deliver 1 μg/mlanti-FcγR antibodies to a target location (e.g., a tissue). In otherspecific embodiments, compositions may contain as little as 0.1 μg mlcross-linked or aggregated IgG.

Anti-FcγR antibodies may be administered in a dose of approximately 1.0μg/mL, in an amount sufficient to deliver 1 μg/ml anti-FcγR antibodiesto the target tissue, or in another dose sufficient to inhibit fibrocyteproliferation or differentiation without causing an undesirable amountof cell death in the patient. Aggregated or cross-linked IgG may beadministered in an amount sufficient to deliver at least 0.1 μg/ml IgGto the target location, or in another dose sufficient to suppressfibrocytes without causing an undesirable amount of cell death in thepatient.

Anti-FcγR antibodies used in examples of the present disclosure includeanti-FcγRI antibodies and anti-FcγRII antibodies. Cross-linked oraggregated IgG may include any IgG able to bind the target FcγR throughits Fc region, provided that at least two such IgG antibodies arephysically connected to one another.

Antibodies of both types may include whole antibodies or a portionthereof, preferably the portion functional in suppressing fibrocytes.For example, they may include any antibody portion able to cross-linkFcγR. This may include aggregated or cross-linked antibodies orfragments thereof, such as aggregated or cross-linked whole antibodies,F(ab′)₂ fragments, and possibly even Fc fragments.

Aggregation or cross-linking of antibodies may be accomplished by anyknown method, such as heat or chemical aggregation. Any level ofaggregation or cross-linking may be sufficient, although increasedaggregation may result in increased fibrocyte suppression. Antibodiesmay be polyclonal or monoclonal, such as antibodies produced fromhybridoma cells. Compositions and methods may employ mixtures ofantibodies, such as mixtures of multiple monoclonal antibodies, whichmay be cross-linked or aggregated to like or different antibodies.

Anti-FcγR antibodies may include any isotype of antibody.

Compositions may be applied locally or systemically. The compositionsmay also be supplied in combinations or with cofactors. Compositions maybe administered in an amount sufficient to restore normal levels, if thecomposition is normally present in the target location, or they may beadministered in an amount to raise levels above normal levels in thetarget location.

The compositions of the present invention may be supplied to a targetlocation from an exogenous source, or they may be made in vivo by cellsin the target location or cells in the same organism as the targetlocation.

Compositions of the present invention may be in any physiologicallyappropriate formulation. They may be administered to an organism byinjection, topically, by inhalation, orally or by any other effectivemeans.

The same compositions and methodologies described above to suppressfibrocytes may also be used to treat or prevent conditions resultingfrom inappropriate fibrocyte proliferation or differentiation. Forexample, they may treat or prevent a condition occurring in the liver,kidney, lung, heart and pericardium, eye, skin, mouth, pancreas,gastrointestinal tract, brain, breast, bone marrow, bone, genitourinary,a tumor, or a wound.

Generally, they may treat or prevent fibrosis resulting from conditionsincluding but not limited to rheumatoid arthritis, lupus, pathogenicfibrosis, fibrosing disease, fibrotic lesions such as those formed afterSchistosoma japonicum infection, radiation damage, autoimmune diseases,Lyme disease, chemotherapy induced fibrosis, HIV or infection-inducedfocal sclerosis, failed back syndrome due to spinal surgery scarring,abdominal adhesion post surgery scarring, fibrocystic formations,fibrosis after spinal injury, surgery-induced fibrosis, mucosalfibrosis, peritoneal fibrosis caused by dialysis, andAdalimumab-associated pulmonary fibrosis.

Specifically, in the liver, they may treat or prevent fibrosis resultingfrom conditions including but not limited to alcohol, drug, and/orchemically induced cirrhosis, ischemia-reperfusion injury after hepatictransplant, necrotizing hepatitis, hepatitis B, hepatitis C, primarybiliary cirrhosis, and primary sclerosing cholangitis.

Relating to the kidney, they may treat or prevent fibrosis resultingfrom conditions including but not limited to proliferative andsclerosing glomerulonephritis, nephrogenic fibrosing dermopathy,diabetic nephropathy, renal tubulointerstitial fibrosis, and focalsegmental glomerulosclerosis.

Relating to the lung, they may treat or prevent fibrosis resulting fromconditions including but not limited to pulmonary interstitial fibrosis,sarcoidosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, asthma,chronic obstructive pulmonary disease, diffuse alveolar damage disease,pulmonary hypertension, neonatal bronchopulmonary dysplasia, chronicasthma, and emphysema. There are several sub-names or synonyms forpulmonary fibrosis including, but not limited to, cryptogenic fibrosingalveolitis, diffuse interstitial fibrosis, idiopathic interstitialpneumonitis, Hamman-Rich syndrome, silicosis, asbestosis, berylliosis,coal worker's pneumoconiosis, black lung disease, coal miner's disease,miner's asthma, anthracosis, and anthracosilicosis.

Relating to the heart and/or pericardium, they may treat or preventfibrosis resulting from conditions including but not limited tomyocardial fibrosis, atherosclerosis, coronary artery restenosis,congestive cardiomyopathy, heart failure, and other post-ischemicconditions.

Relating to the eye, they may treat or prevent fibrosis resulting fromconditions including but not limited to exophthalmos of Grave's disease,proliferative vitreoretinopathy, anterior capsule cataract, cornealfibrosis, corneal scarring due to surgery, trabeculectomy-inducedfibrosis, progressive subretinal fibrosis, multifocal granulomatouschorioretinitis, and other eye fibrosis.

Relating to the skin, they may treat or prevent fibrosis resulting fromconditions including but not limited to Depuytren's contracture,scleroderma, keloid scarring, psoriasis, hypertrophic scarring due toburns, atherosclerosis, restenosis, and psuedoscleroderma caused byspinal cord injury.

Relating to the mouth and/or esophagus, they may treat or preventfibrosis resulting from conditions including but not limited toperiodontal disease scarring, gingival hypertrophy secondary to drugs,and congenital esophageal stenosis.

Relating to the pancreas, they may treat or prevent fibrosis resultingfrom conditions including but not limited to pancreatic fibrosis,stromal remodeling pancreatitis, and stromal fibrosis.

Relating to the gastrointestinal tract, they may treat or preventfibrosis resulting from conditions including but not limited tocollagenous colitis, villous atrophy, cryp hyperplasia, polyp formation,fibrosis of Crohn's disease, and healing gastric ulcer.

Relating to the brain, they may treat or prevent fibrosis resulting fromconditions including but not limited to glial scar tissue.

Relating to the breast, they may treat or prevent fibrosis resultingfrom conditions including but not limited to fibrocystic disease anddesmoplastic reaction to breast cancer.

Relating to the bone marrow, they may treat or prevent fibrosisresulting from conditions including but not limited to fibrosis inmyelodysplasia and neoplastic diseases.

Relating to the bone, they may treat or prevent fibrosis resulting fromconditions including but not limited to rheumatoid pannus formation.

Relating to the genitourinary system, they may treat or prevent fibrosisresulting from conditions including but not limited to endometriosis,uterine fibroids, ovarian fibroids, and Peyronie's disease.

Relating to radiation induced damage, they may treat or prevent fibrosisrelated to, but not limited to, treatment of head and neck cancer,ovarian cancer, prostate cancer, lung cancer, gastrointestinal cancer,colon cancer, and breast cancer.

The following examples are included to demonstrate specific embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples that follow representtechniques discovered by the inventors to function well in the practiceof the invention. However, those of skill in the art should, in light ofthe present disclosure, appreciate that many changes can be made in thespecific embodiments that are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention.

EXAMPLES Example 1 Fibrocyte Differentiation Assay

Human peripheral blood mononuclear cells (PBMC) were isolated from buffycoats (Gulf Coast Regional Blood Center, Houston, Tex.) by Ficoll-PaquePlus (Amersham Biosciences, Piscataway, N.J.). Cells were incubated inserum-free medium (SFM), which consists of RPMI (Invitrogen, Carlsbad,Calif.) supplemented with 10 mM HEPES (Invitrogen), 2 mM glutamine, 100U/ml penicillin, 100 μg/ml streptomycin, and 1×ITS-3 (500 μg/ml bovineserum albumin, 10 μg/ml insulin, 5 μg/ml transferrin, 5 ng/ml sodiumselenite, 5 μg/ml linoleic acid, and 5 μg/ml oleic acid; Sigma-Aldrich,St. Louis, Mo.). Normal human serum (Sigma-Aldrich) was added at 1%.PBMC were cultured in flat-bottomed 96 well tissue culture plates (Type353072, BD Biosciences Discovery Labware, Bedford, Mass.) in 200 μlvolumes at 2.5×10⁵ cells per ml in a humidified incubator containing 5%CO₂ at 37° C. for 5 days. Fibrocytes were identified by morphology inviable cultures as adherent cells with an elongated spindle-shapedmorphology as distinct from lymphocytes or adherent monocytes.Enumeration of fibrocytes was performed on cells cultured for 5 days.Cells were air dried, fixed in methanol and stained with eosin andmethylene blue (Hema 3 Stain, Fisher Scientific, Hampton, N.H.).Fibrocytes from duplicate wells were counted in five different 900 μmdiameter fields per well, using the above criteria of an elongatedspindle-shape and the presence of an oval nucleus. All cultures werecounted by at least two independent observers. The number of fibrocytesobserved was 1.2±0.6×10⁴ (mean±SD, n=12 healthy individuals) fibrocytesper ml of peripheral blood, with a range of 3.7×10³ to 2.9×10⁴fibrocytes per ml. These results indicate that fibrocyte precursorsaccount for approximately 1% of the total peripheral blood mononuclearcells.

Example 2 Antibodies, Proteins, and Inhibitors

Human IgA, IgG, IgM, and IgG F(ab′)₂ fragments were from JacksonImmunoResearch Laboratories, West Grove, Pa. Goat F(ab′)₂ anti-humanIgG, goat F(ab′)₂ anti-murine IgG, goat F(ab′)₂ anti-rabbit IgG, andwhole mouse IgG1, whole mouse IgG2a and mouse F(ab′)₂ IgG1 isotypecontrol antibodies were from Southern Biotechnology Associates Inc.,Birmingham, Ala. Sheep red blood cells (SRBC) and rabbit anti-SRBC werefrom ICN, Irvine, Calif. F(ab′)₂ fragments of the blocking monoclonalantibodies to FcγRI (clone 10.1, IgG1 isotype) and FcγRII (clone 7.3,IgG1 isotype) were from Ancell, Bayport, Minn. The following primarymonoclonal antibodies were used for immunohistochemistry: anti-CD14(clone M5E2, IgG2a, BD-Biosciences, San Diego, Calif.), anti-CD34 (cloneQBend10, IgG1, GeneTex, San Antonio, Tex.), CD 43 (clone IG10, IgG1,BD), pan-CD45 (clone H130, IgG1, BD), anti-prolyl 4-hydrolase (clone5B5, IgG1, Dako, Carpinteria, Calif.), and anti-alpha smooth muscleactin (clone 1A4, IgG2a, Sigma-Aldrich, St. Louis, Mo.). Collagen-I wasdetected using an affinity-purified rabbit polyclonal antibody fromRockland, Gilbertsville, Pa. PP2 (AG 1879;4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), PP34-Amino-7-phenylpyrazol[3,4-d]pyrimidine) and the Syk inhibitor(3-(1-Methyl-1H-indol-3-yl-methylene)-2-oxo-2,3-dihydro-1H-indole-5-sulfonamide)were from Calbiochem, EMD Biosciences, San Diego, Calif.

Example 3 Inhibition of Fibrocyte Differentiation

To determine if anti-FcγR antibodies activate FcγR to inhibit fibrocytedifferentiation, PBMC at 2.5×10⁵ per ml were cultured in serum-freemedium for 5 days in the presence or absence of 1 μg/ml of free orcross-linked F(ab′)₂ antibodies to FcγRI or FcγRII.

To crosslink individual FcγR, PBMC were incubated for 30 minutes at 4°C. with 1 μg/ml F(ab′)₂ anti-FcγRI or F(ab′)₂ anti-FcγRII, and receptorswere then cross-linked by the addition of 500 ng/ml F(ab′)₂ goatanti-mouse IgG for 30 minutes at 4° C. PBMC were then warmed to 37° C.and cultured for 5 days.

After the cells were cultured in the presence or absence of free orcross-linked F(ab′)₂ antibodies to FcγRI or FcγRII, the cells were thenair-dried, fixed, stained, and fibrocytes were enumerated by morphology.The results of this example are shown in FIG. 1.

Compared to PBMC cultured in the presence of 500 ng/ml goat F(ab′)₂anti-mouse IgG alone, cells cultured in the presence of 500 ng/ml goatF(ab′)₂ anti-mouse IgG and anti-FcγRI or anti-FcγRII significantlyinhibited fibrocyte differentiation (p<0.01, indicated by **), asdetermined by ANOVA (n=3 separate donors). These results suggest thatcross-linking either FcγRI or FcγRII alone significantly inhibitedfibrocyte differentiation. However, there was no additional inhibitionwhen both receptors were cross-linked together, suggesting that nosynergistic interaction occurs (FIG. 1). These experiments show thatfibrocyte differentiation can be inhibited to approximately 50% by theaddition of 1 μg/ml anti-FcγR. Greater inhibition could be achieved byincubating PBMC with higher concentrations of anti-FcγR (5 and 10μg/ml), however these concentrations of anti-FcγR also led tosignificant cell death (data not shown). These results suggest thatligation and cross-linking of FcγRI or FcγRII can inhibit fibrocytedifferentiation.

Although PBMC may contain various cells that may form fibrocytes,including monocytes, monocyte cultures alone may also differentiate toform fibrocytes. This phenomenon is show in FIG. 3. FIG. 3 alsoindicates that this fibrocyte differentiation is suppressed by theaggregation of FcγR. Specifically, compared to monocytes cultured inserum free medium (SFM), aggregated human IgG was able to cross-linkFcγR through its Fc region significantly inhibited fibrocytedifferentiation (p<0.01), as determined by ANOVA.

Example 4 Inhibition of Fibrocyte Differentiation is SYK- and SRC KinaseDependent

FcγR activation leads to a cascade of signaling events initiated by twomain kinases. The initial events following FcγR aggregation involve thephosphorylation of intracellular immunoreceptor tyrosine activationmotifs (ITAM) present on the cytoplasmic tail of FcγRII or the FcRγchain associated with FcγRI, by src-related tyrosine kinases (SRTK). Inmonocytes, the main src-kinases associated with FcγRI and FcγRII are hckand lyn. The phosphorylated ITAM then recruits cytoplasmicSH2-containing kinases, especially Syk, to the ITAMs and Syk thenactivates a series of downstream signaling molecules.

To determine the roles of SRTK and Syk in the regulation of fibrocytedifferentiation, PBMC were pre-incubated with the specific SRTKinhibitor PP2, PP3 as a control for PP2, or the specific Syk inhibitor3-(1-methyl-1H-indol-3-yl-methylene)-2-oxo-2,3-dihydro-1H-indole-5-sulfonamide,before the addition of anti-FcγR antibodies. This Syk inhibitor was usedinstead of the standard Syk inhibitor piceatannol, as piceatannol atconcentrations used to inhibit Syk in whole cells (10 μM) also inhibitsa variety of other enzymes and transcription factors. These proteinsinclude the catalytic subunit of protein kinase A, protein kinase C,myosin light chain kinase, TNF-induced NF-kB activation, and interferonα-mediated signaling via STAT proteins.

Inhibition of fibrocyte differentiation by activating either FcγRI orFcγRII alone or both receptors together was dependent on STRK and Syk,as the inhibition was lost when PBMC were pre-incubated with either PP2or the Syk inhibitor (FIG. 2). Compared to control cultures or culturesincubated with of 500 ng/ml goat F(ab′)₂ anti-mouse IgG (X-linker only),PBMC cultured with 500 ng/ml goat F(ab′)₂ in addition to anti-mouse IgGanti-FcγRI, anti-FcγRII or both antibodies significantly inhibitedfibrocyte differentiation (p<0.05), as determined by ANOVA. The presenceof PP2 or Syk inhibitor, but not the control compound PP3, inhibitedthis inhibition. These data suggest that anti-FcγR antibodies inhibitfibrocyte differentiation through a pathway involving both Syk and SRTK.

Similar results were found when monocyte samples, rather than PBMC wereused to perform tests. Specifically, in FIG. 3, compared to monocytesincubated with 10 μg/ml aggregated human IgG (able to bind to FcγRthrough its Fc region), pre-incubation with PP2 (p<0.01) or Sykinhibitor (p<0.05) significantly inhibited the ability of IgG to inhibitfibrocyte differentiation as determined by ANOVA.

Example 5 IgG Immune Complexes Inhibit Fibrocyte Differentiation

In addition to FcγR, monocytes express IgA receptors, low numbers of IgEreceptors, and the recently characterized IgM receptor. To determine ifother immunoglobins inhibit fibrocyte differentiation, native orheat-aggregated IgA, IgE, IgG or IgM were added to PBMC. The results ofthis example are shown in FIG. 4C. Only heat-aggregated IgG, but notmonomeric IgG or monomeric or heat-aggregated IgA, IgE or IgM, couldinhibit fibrocyte differentiation. This suggests that ligation andcross-linking of FcγR receptors is an inhibitory signal for fibrocytedifferentiation, but that ligation of the other immunoglobin receptorshas no effect on fibrocyte differentiation.

Example 6 Cross-Linked IgG Inhibits Fibrocyte Differentiation

PBMC were incubated with the indicated various concentrations ofmonomeric human IgG for 60 minutes. PBMC were then washed and incubatedin the presence or absence of 500 ng/ml goat F(ab′)2 anti-human IgG.PBMC were then cultured at 2.5×10⁵ cells per ml in serum-free medium for5 days. PBMC were then air-dried, fixed, stained, and fibrocytes wereenumerated by morphology. Results are shown in FIG. 4A. Specifically,compared to monomeric IgG, cross-linked human IgG clearly inhibitedfibrocyte differentiation as compared to non-cross-linked IgG at 0.1μg/ml. At 10 and 100 μg/ml inhibition of differentiation was significant(p=0.03 and p=0.003, respectively, as determined by Student's t test.Additional experiments using sheep red blood cells (SRBC) eitheropsinized or not opsinized with rabbit anti-SRBC IgG indicated that theopsinized SRBC significantly inhibited fibrocyte differentiation(p=0.018) (data not shown).

PBMC were also cultured as above in the presence of the indicatedconcentrations of heat-aggregated human IgG or heat-aggregated humanF(ab′)₂. Results of this example are shown in FIG. 4B. Compared toheat-aggregated human F(ab′)₂, heat-aggregated whole IgG significantlyinhibited fibrocyte differentiation at concentrations of 25 μg/ml andhigher, as determined by Student's t test.

Although only exemplary embodiments of the invention are specificallydescribed above, it will be appreciated that modifications andvariations of these examples are possible without departing from thespirit and intended scope of the invention.

Example 7 Antibody Studies

SAP and CRP augment phagocytosis and bind to Fcγ receptors on a varietyof cells. CRP binds with a high affinity to FcγRII (CD32), a loweraffinity to FcγRI (CD64), but does not bind FcγRIII (CD16). SAP binds toall three classical Fcγ receptors, with a preference for FcγRI andFcγRII. Monocytes constitutively express FcγRI. Because this receptorbinds monomeric IgG, it is saturated in vivo. In order to determinewhether the presence of monomeric human IgG could prevent SAP frominhibiting fibrocyte differentiation, PBMC were cultured in serum-freemedium in the presence of a range of concentrations of monomeric IgG for60 minutes. SAP, at the concentrations indicated in FIG. 5, was thenadded and the cells were cultured for 4 days. As described in the aboveexamples, 2.5 μg/ml SAP in the absence of IgG strongly inhibitedfibrocyte differentiation. (See FIG. 5.) Monomeric IgG in a range from0.1 to 1000 μg/ml, which corresponds to approximately 0.001 to 10% serumrespectively, had little effect on the suppression of fibrocyteformation by SAP.

To determine whether ligation and cross-linking of Fc receptors couldalso influence monocyte to fibrocyte differentiation, three test sampleswere used; soluble immune complexes (ovalbumin-antibody), particulateimmune complexes, including opsonised SRBC and heat-aggregated IgG. PBMCcultured for 4 days with ovalbumin or anti-ovalbumin mAb showed that thetwo proteins alone had a modest effect on the differentiation ofmonocytes compared to cultures where no reagent was added. (See FIG.6A.) However, the addition of ovalbumin:anti-ovalbumin immune complexesled to a significant reduction in the number of differentiatedfibrocytes (See FIG. 6A). A similar effect was observed when PBMC werecultured with opsonised SRBC. SRBC opsonised with rabbit anti-SRBC at20:1 and 40:1 SRBC:monocyte ratios significantly suppressed fibrocytedifferentiation as compared to cells cultured with SRBC alone (See FIG.6B). Finally, PBMC cultured with heat-aggregated IgG, but notheat-aggregated F(ab)₂, also showed potent inhibition of fibrocytedifferentiation (See FIG. 6C.) Together these data suggest that ligationand cross-linking of Fc receptors is suppressor of monocyte to fibrocytedifferentiation.

The observation that immune complexes inhibit fibrocyte differentiationsuggests that one or more FcγR influences fibrocyte differentiation. Toexamine the role of FcγR in fibrocyte differentiation PBMC were culturedin the presence or absence of blocking antibodies to FcγRI (CD64),FcγRII (CD32) or FcγRIII (CD16) before the addition of SAP, or as acontrol CRP. When samples were pre-incubated with a blocking mAb foreach of the three FcγR, SAP was later able to modestly suppressfibrocyte differentiation. However, in the absence of exogenously addedSAP, the FcγRI (CD64) blocking mAb had a profound effect on fibrocytedifferentiation. Incubation of PBMC with blocking mAb to FcγRI, but notFcγRII or FcγRIII, promoted fibrocyte differentiation as compared tocells cultured with isotype-matched control mAb or cells cultured withno mAb (P<0.01) (See FIG. 7). These data suggested that SAP or IgG,might have been produced by some cells in the culture system over 4days, or that SAP or IgG was retained by cells from the blood. Westernblotting failed to show the presence of SAP or IgG after cells had beencultured for 4 days in vitro. This suggests that the FcγRI blocking mAbhas a direct effect on fibrocyte differentiation or that SAP or IgG wereonly present during the early time points of the cell culture.

1. A method of suppressing fibrocyte formation in a subject in needthereof comprising administering to the subject having pulmonaryfibrosis an anti-FcγR antibody in an amount sufficient to suppressfibrocyte formation in a lung.
 2. The method of claim 1, wherein theanti-FcγR antibody is administered at a concentration of at least 1.0μg/ml.
 3. The method of claim 1, wherein the anti-FcγR antibody isadministered at a concentration of at least 0.1 μg/ml.
 4. The method ofclaim 1, wherein the antibody is an IgG.
 5. The method of claim 1,wherein the antibody is an anti-FcγRI antibody.
 6. The method of claim1, wherein the antibody is an anti-FcγRII antibody.
 7. The method of anyone of claims 4-6, wherein the antibody comprises an F(ab′)₂ fragment.8. The method of any one of claims 4-6, wherein the antibody comprisesan Fc fragment.
 9. The method according to claim 1, wherein thepulmonary fibrosis comprises a condition selected from the groupconsisting of: Adalimumab-associated pulmonary interstitial fibrosis,sarcoidosis, idiopathic pulmonary fibrosis, asthma, chronic obstructivepulmonary disease, diffuse alveolar damage disease, pulmonaryhypertension, neonatal bronchopulmonary dysplasia, and emphysema. 10.The method of claim 1, wherein the antibody is an anti-FcγRIII antibody.